BackgroundIn humans, it is now well documented that rising paternal age is correlated with decreased sperm DNA integrity and embryonic developmental failures. On the other side of the coin, it is also reported that very young fathers such as teenagers carry an increased risk of adverse birth outcomes. These observations suggest that, at least in humans, there is an age window for optimal sperm DNA integrity. In bovine, little is known about sperm DNA quality in young bulls and how it evolves with age. This study aimed to fill in this gap as it may be of importance for the bovine industry to know when exactly a bull is an optimal performer for reproductive programs.MethodsForty Nellore bulls were divided into three age groups: 1.8 to 2 years – young bulls; 3.5 to 7 years – adult bulls; and 8 to 14.3 years – aged bulls. Three ejaculates were collected from each bull, cryopreserved and evaluated for various parameters including: computer-assisted sperm analysis (CASA), plasma membrane and acrosome integrity, mitochondrial potential, sperm nuclear protamination, DNA oxidative damage, and Sperm Chromatin Structure Assay (SCSA).ResultsWe report here that young bulls presented superior values for motility, plasma and acrosomal membrane integrity, and high mitochondrial potential. However, they also presented higher values for sperm morphological abnormalities compared to adult and aged animal groups (p < 0.05). In addition, young bulls exhibited more defective protamination than older animals did. The oldest bulls showed more nuclear oxidative damage than the younger groups of bulls while both the young and aged groups were found more susceptible to DNA denaturation as revealed with the SCSA test (p < 0.05).ConclusionThese results indicate that young bulls spermatozoa best survived the freezing procedure, followed by adult and aged bulls. However, young and aged bulls were found to be more susceptible to DNA damage, respectively caused by protamine deficiency and oxidation. Therefore, although young bulls have correct semen parameters according to classical evaluation, our results indicate that they may show some structural nuclear immaturity.

The aim of this study was to evaluate the chromatin packing and sperm head morphometry of cryopreserved semen of Nelore bulls (Bos taurus indicus) of different ages. Furthermore, the influence of the degree of chromatin compaction on in vitro embryo production (IVP) was investigated. Forty bulls were divided into three groups: young (1.8-2 years), adult (3.5-7 years), and senile (8-14.3 years). The ejaculates were frozen according to standards established by the Artificial Insemination Center located in the Southeast of Brazil. Toluidine blue staining was used for simultaneous evaluation of the sperm chromatin and sperm head morphometry. Chromomycin A3 (CMA3) was applied to analyze sperm protamination and IVP for embryonic development. Spermatozoa of young bulls presented higher values for area (A, pixels), perimeter (P, pixels), and width (W, pixels) compared to adults and senile (young: A = 1848.5 ± 119.79, P = 10.23 ± 0.29, and W = 1.95 ± 0.1; adults: A = 1672.9 ± 104.46, P = 9.86 ± 0.33, and W = 1.81 ± 0.06; senile: A = 1723.1 ± 124.41, P = 9.97 ± 0.33, and W = 1.83 ± 0.09; P < 0.0001) and showed higher protamination deficiency when analyzed by CMA3 (young: 1.57 ± 0.76; adults: 1.09 ± 0.63, and senile: 0.90 ± 0.59; P < 0.05). Likewise, variables of sperm head size (A, P, and W) and protamination assessed by CMA3 showed negative correlation with age and positive correlation with ellipticity, evaluated by toluidine blue method (P < 0.05). Sperm head area was larger in spermatozoa presenting chromatin instabilities than spermatozoa without chromatin alteration (P < 0.0001). There was no difference in IVP when using semen with larger or smaller portions of spermatozoa with chromatin instabilities, indicating that the proportion of sperm with abnormal chromatin compaction (4%-16.15%) did not interfere with early embryonic development. From our results, it can be concluded that sperm of young Nelore bulls have larger heads compared to adults and senile due to reduced protamine content when evaluated by CMA3 and higher proportion of major sperm defects assessed by differential interference contrast microscopy.

The routine semen evaluation assessing sperm concentration, motility and morphology, does not identify subtle defects in sperm chromatin architecture. Bulls appear to have stable chromatin, with low levels of DNA fragmentation. However, the nature of fragmentation and its impact on fertility remain unclear and there are no detailed reports characterizing the DNA organization and damage in this species. The intensive genetic selection, the use of artificial insemination and in vitro embryo production associated to the cryopreservation process can contribute to the chromatin damage and highlights the importance of sperm DNA integrity for the success of these technologies. Frozen-thawed semen samples from three ejaculates from a Nellore bull showed high levels of morphological sperm abnormalities (55.8±5.1%), and were selected for complementary tests. Damage of acrosomal (76.9±8.9%) and plasma membranes (75.7±9.3%) as well as sperm DNA strand breaks (13.8±9.5%) and protamination deficiency (3.7±0.6%) were significantly higher compared to the values measured in the semen of five Nellore bulls with normospermia (24.3±3.3%; 24.5±6.1%; 0.6±0.5%; 0.4±0.6% for acrosome, plasma membrane, DNA breaks and protamine deficiency, respectively) (P<0.05). Motility and percentage of spermatozoa with low mitochondrial potential showed no differences between groups. This study shows how routine semen analyses (in this case morphology) may point to the length and complexity of sperm cell damage emphasizing the importance of sperm function testing.

The aims of this study were to evaluate cryopreserved semen of Nellore bulls of different ages and verify whether sperm quality declines with advancing age and whether lipid peroxidation and DNA damage are involved in this process. For this purpose, 40 Nellore bulls were divided into three age groups: Young, aged 1.8-2 years (n = 9); Adult, aged 3.5-7.0 years (n = 19); and Seniors, aged 8.0-14.3 years (n = 12). Three ejaculates were collected from each bull, cryopreserved and evaluated for various parameters including membrane integrity, mitochondrial potential (FITC-PSA and JC1), lipid peroxidation (C-11BODIPY 581 / 591) and oxidative DNA damage (8OHdG) using flow cytometry. The thawed semen of senior bulls was characterized by a low percentage of motile sperm (33.7 ± 6.1%), higher damage to the plasma and acrosomal membrane (37.5 ± 9.8%), and low mitochondrial potential (29.1 ± 13.8%), as well as higher percentages of peroxidated cells (53.6 ± 12.2%) and DNA damage (44.1 ± 11.0%; P < 0.05). Lipid peroxidation was negatively correlated with motility (r = -0.35, P < 0.0002), average mitochondrial potential (r = -0.42; P < 0.0001) and showed a positive correlation with membrane injury and oxidative DNA damage (r = 039; P = 0.0003). Young bulls presented superior thawed sperm quality, possibly due to greater resistance to oxidative stress and, consequently, to cryopreservation. In conclusion, the sperm quality of bull semen declines with advancing age and is strongly associated with increased oxidative damage to both the plasma membrane and DNA.

Background: Seminomas are germ cell tumors mainly originating from spermatogonia in the seminiferous tubules and has been described in several domestic animal species, even though has rarely been described in goats. Moreover, seminomas tumors are not usually metastatic and rarely trigger paraneoplastic syndrome. In this sense, this is a case report of a 12-year-old male Alpine goat, suspected of testicular neoplasm based on clinical examination and ultrasound imaging, diagnosed as a seminoma with metastasis in liver by histopathology upon necropsy. Case: A 12-year-old male Alpine goat presented a 10 month history of progressive weight loss, prostration, and scrotal sac enlargement. The major clinical findings were pale conjunctival mucus, bilateral nasal catarrhal secretion, obstructive dyspnea, an increased abdominal component, crackling at trachea auscultation, silence upon lung auscultation of the ventral area and wheezing upon auscultation of the dorsal area, and enlargement of the left testicle with contralateral atrophy. At the Ultrasonography scan, the enlarged left testicle presented architecture loss, as well as circumscribed masses differing in echogenicity and echotexture with scattered small hyperechoic nodules. The shrunken right testicle showed acoustic shading across the surface suggestive of calcification. Due to the poor prognosis and regard for animal welfare, the goat was euthanized. The main necropsy findings on testicles were: enlarged left testicle with white parenchyma on the dorsal side as well as diffuse yellow elliptical lesions of 0.5-2.5 cm on the surface in association with two circumscribed areas at the cranial and caudal poles, firm upon cutting. The right testicle was half the typical size, slightly pale, firm at cutting of the tunica albuginea, and presented dark parenchyma with abundant calcification dots suggestive of microlithiasis. The histological findings included diffuse tumoral stroma of the left testicle composed of large, polyhedral, discretely demarcated circular cells exhibiting a large nucleus varying in size with little cytoplasm and a high mitotic rate. The right testicle exhibited areas of necrosis and fibrosis of the testicular parenchyma, contiguous with the area of degeneration and normal parenchyma, caseous necrosis, and focal calcification. The liver presented some foci of the same cellular pattern of the seminoma, as described in the parenchyma. The findings summarized at necropsy and histopathological evaluation were seminoma in the left testicle, with metastasis to the liver; degeneration and necrosis of the right testicle with abscess and calcification. Discussion: Seminoma has been described in several species, even though rarely in goats. This report was the second ever seminoma reported in caprine species, and particularly this case presented liver metastasis, showing the pathologic potential of this neoplasia for this species, and it differed from first reported which described ulcerative lesions in, and adhesion of the skin covering the testis. Regard...